The objective of this research proposal is to develop a convenient, generic methodology to modulate the physico-chemical properties of proteins by fusing them to environmentally-responsive elastin-like polypeptides (ELPs), and to concurrently elucidate the biophysical principles which govern modulation of their properties. The underlying hypothesis of the proposed research is that incorporation of an ELP sequence at the N- or C-terminus of a target protein will impart environmentally-responsive properties to the fusion protein. This is because ELPs are oligomeric repeats of the pentapeptide sequence Val-Pro-Gly-X-Gly (VPGXG)(X is any amino acid except Pro), which undergo an "inverse" phase transition: below the inverse transition temperature [T(t)] ELPs are soluble in aqueous solution, but when the temperature is raised above their T(t), they undergo a sharp (2-3 degree C range) phase transition, leading to desolvation and aggregation of the polypeptide. The inverse transition can be induced by changes in temperature, ionic strength, or pH, and is completely reversible. In preliminary studies, Dr. Chilkoti has demonstrated that the solution and interfacial properties of ELP fusion proteins can be systematically modulated as a function of their solution environment (e.g., temperature and ionic strength). He has also shown that the inverse transition of an ELP in a fusion protein is related to the effective surface hydrophobicity (ESH) of the fusion partner. Dr. Chilkoti proposes to synthesize a set of ELP fusion proteins in which ESH of the protein and MW of the ELP is independently varied. He will experimentally determine the altered T(t), of ELP fusion proteins relative to ELP control [delta T(t)], and ESH of the fusion proteins, and investigate the relationship between delta T(t) and ESH. The proposed research will result in a fundamental biophysical understanding of the parameters which govern modulation of the inverse transition of ELP fusion proteins, which will allow rational design of parameters (e.g., temperature range, ELP MW, ionic strength) for the proposed biomolecular engineering applications of ELP fusion proteins. Specific applications that will be developed in this proposal are: (1) inverse transition cycling, a new, and convenient methodology for protein purification based upon thermally-reversible modulation of the solubility of ELP fusion proteins; and (2) biosensor regeneration, which utilizes the thermally-reversible adsorption of ELP fusion proteins on hydrophobic surfaces.